UNIVERSITI PUTRA MALAYSIA EFFECTS OF ETHANOLIC EXTRACT OF COCOA ON BLOOD GLUCOSEAND LIPID PROFILE IN STREPTOZOTOCIN-INDUCED DIABETIC RATS RUZAIDI AZLI BIN MOHD MOKHTAR FPSK(M) 2005 27
EFFECTS OF ETHANOLIC EXTRACT OF COCOA ON BLOOD GLUCOSE AND LIPID PROFILE IN STREPTOZOTOCIN-INDUCED DIABETIC RATS RUZAIDI AZLI BIN MOHD MOKHTAR MASTER OF SCIENCE UNIVERSITI PUTRA MALAYSIA
EFFECTS OF ETHANOLIC EXTRACT OF COCOA ON BLOOD GLUCOSE AND LIPID PROFILE IN STREPTOZOTOCIN-INDUCED DIABETIC RATS BY RUZAIDI AZLI BIN MOHD MOKHTAR Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia, in Fulfillment of the Requirements for the Degree of Master of Science April 2005
%is 17iRFi.s is specially dedicated to my belbved For th unconditionalpatient, Lhve and support.
Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfilment of the requirement for the degree of Master of Science EFFECTS OF ETHANOLIC EXTRACT OF COCOA ON BLOOD GLUCOSE AND LIPID PROFILE IN STREPTOZOTOCIN INDUCED DIABETIC RATS BY RUZAIDI AZLI BIN MOHD MOKHTAR April 2005 Chairman: Faculty: Amin Ismail, PhD Medicine and Health Sciences This study aims to investigate the hypoglycaemic and hypocholesterolaemic properties of Malaysian cocoa (Theobroma cacao) polyphenols extract in-vivo and in-vitro. Cocoa extract (contained 190-286 mg total polyphenol per g of extract) was prepared from fermented and roasted (140 "C, 20 min) beans by extracting with 80% ethanol in the ratio of 1 to 10. The total phenolic content was estimated according to the Folin-Ciocalteu reagent method. The eluted individual polyphenol was monitored by using a normal-phase HPLC. Monomer is the predominant polyphenols present in cocoa extract (CE) followed by dimer and tetramer. To study the effect of CE on plasma glucose levels and lipid profiles in normal and diabetic rats, two different batches of animal (in-vivo) studies were performed. In the first batch, rats were given free excess to diet containing CE in the form of powder, while in the second batch, rats were force-fed with CE suspended in normal saline daily. The CE was given in three dosages (100, 200 and 300 mg per kg body weight) to both batches for a period of 4 weeks. The result showed that 100 mgkg and 300
mgkg CE significantly reduced (p < 0.05) the plasma glucose levels in the diabetic rats of both the first and second batch of studies. In the first batch, supplementation of 100 mgkg and 300 mglkg CE had significantly reduced (p < 0.05) the level of total cholesterol in diabetic rats. In addition, 100, 200 and 300 mgkg CE diets had significantly lowered (p < 0.05) the total triglycerides. Interestingly, this study found that plasma HDL-cholesterol had increased significantly (p < 0.05) in diabetic rats fed with 200 mgkg CE, while the LDL-cholesterol had decreased significantly (p < 0.05) in group treated with 100 mgkg CE. In the second batch, plasma cholesterol, HDL-cholesterol and LDL-cholesterol levels showed no siginificant difference in both normal and diabetic rats. Meanwhile, there was a significant decrease (p < 0.05) in plasma triglyceride level in diabetic rats. In another study, rats were pretreated with CE to investigate the protective effect of CE against streptozotocin diabetogenic action. In 200 mglkg CE pretreated rats, there was a 163% increase in plasma glucose levels, compared with a 226% increase in diabetic control rats. There were no protective effects on plasma lipid profiles in CE pretreated rats. Results also exhibited CE could normalize the body weight loss caused by STZ. BRIN-BD11 cell lines (in-vitro) were used to evaluate the effect of CE on insulin secretion. This invitro study demonstrated that CE at a concentration of 0.1 mglrnl significantly increase (p < 0.05) insulin secretion compared to control. In conclusion, the study shows that Malaysian cocoa polyphenol extract may possess potential hypoglycaernic and hypochlosterolaernic properties. Further studies are needed to elucidate the exact mechanism by which polyphenols present in CE can lower the plasma glucose levels and improved lipid profiles in diabetic rats, and stimulate insulin secretion in BRIN-BDl1 cell lines.
Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai memenuhi keperluan untuk ijazah Master Sains KESAN EKSTRAK ETANOLIK KOKO KEATAS GLUKOSA DARAH DAN PROFAIL LIPID TIKUS DIABETES DIARUH STREPTOZOTOCIN - Pen~erusi: Amin Isrnail, PhD Oleh RUZAIDI AZLI BIN MOHD MOKHTAR April 2005 Fakulti: Perubatan dan Sains Kesihatan Kajian ini bertujuan untuk mengkaji ciri-ciri hipoglisemik dan hipokolesterolemik ekstrak polifenol koko Malaysia (Theobroma cacao) secara in-vivo dan in-vitro. Ekstrak koko (mengandungi 190-286 mg polifenol per g ekstrak) disediakan daripada biji koko yang telah difermentasi dan dipanggang (140 "C, 20 min) dengan mengekstrak menggunakan 80% etanol berdasarkan nisbah 1 kepada 10. Pengiraan jumlah kandungan polifenol menggunakan kaedah Folin-Ciocalteu. Polifenol individu ditutdisis dengan menggunitkan fsa normal HPLC. Moncmw addah polifenol yang paling banyak terdapat di dalam CE diikuti oleh dimer dan tetrarner. Untuk mengkaji kesan CE ke atas paras plasma glukosa dan profil lipid tikus normal dan diabetik, dua kumpulan kajian haiwan (in-vivo) telah dijalankan. Dalam kajian kumpulan pertama, tikus tersebut bebas untuk mengambil makanan dalam bentuk serbuk yang mengandungi CE, sementara dalam kumpulan kedua, tikus diberi CE yang dilarutkan di dalam salin normal secara oral (suapan paksa). CE diberi dalam tiga dos (100, 200 dan 300 mg per kg berat badan), dan diberi kepada kedua-dua
kumpulan selama 4 rninggu. Keputusan menunjukkan 100 mgkg dan 300 mgkg CE menurunkan paras plasma glukosa secara signifikan (p < 0.05) di dalarn kedua-dua kumpulan. Di dalam kajian kumpulan pertama, pemberian 100 mgkg dan 300 mgkg CE menurunkan paras plasma kolesterol secara signifikan (p < 0.05) di dalam tikus diabetik. Tambahan pula, 100, 200 dan 300 mgkg diet CE menurunkan kandungan trigliserida secara signifikan (p < 0.05). Kajian ini juga mendapati plasma HDLkolesterol meningkat secara signifikan (p < 0.05) di dalam tikus diabetik yang diberi 200 mgkg CE, sementara LDL-kolesterol menurun secara signifikan di dalam tikus diberi 200 mgkg CE. Di dalam kajian kumpulan kedua, paras plasma kolesterol, HDL-kolesterol dan LDL-kolesterol tidak menunjukkan perubahan yang signifikan di dalam tikus normal dan diabetik. Sementara itu, terdapat penurunan signifikan (p < 0.05) di dalam paras plasma trigliserida tikus diabetik. Untuk mengkaji kesan perlindungan CE melawan tindakan diabetogenik STZ, tikus diberi CE terlebih dahulu sebelum suntikan STZ. Tikus yang terlebih dahulu diberi 200 mgkg CE, didapati terdapat peningkatan 163% paras plasma glukosa, berbanding dengan peningkatan 226% di dalam tikus kontrol diabetik. Tiada kesan perlindungan ke atas plasma profil lipid di dalam tikus yang diberi CE terlebih dahulu. Keputusan juga mendapati CE berupaya mengnormalkan kehilangan berat badan disebabkan oleh STZ. BRIN-BD 11 sel (in-vitro) digunakan untuk menilai kesan CE ke atas rembesan insulin. Di dalarn kajian ini menunjukkan CE pada kepekatan 0.1 mghl meningkatkan rembesan insulin secara signifikan (p < 0.05) berbanding kontrol. Secara kesimpulan, kajian ini menunjukkan ekstrak polifenol koko Malaysia kemungkinan mempunyai potensi ciri-ciri hipoglisemik dan hipokolesterolemik. Kajian selanjutnya diperlukan untuk menerangkan mekanisme keupayaan polifenol
daripada CE dalam menurunkan paras plasma glukosa dan memperbaiki profil lipid di dalam tikus diabetik, dan merangsang rembesan insulin di dalam BRIN-BDll sel. vii
ACKNOWLEDGEMENTS In the name of Allah, most gracious, most merciful. Alharndulillahirabbil'alarnin, thanks to Allah for giving me the time and courage in completing this project. First and foremost, I would like take this oppurtinity to express my sincere appreciation and heartfelt gratitude to my project supervisor, Dr. Amin Ismail, for his invaluable guidance, encouragement, advice, support and also his endless patience in giving me suggestions and corrections in making this project a success. I am also grateful to the members of my supervisory committee, Dr. Muhajir Hamid and Pn. Nawalyah Abdul Ghani for their constructive comments and suggestions. I'm sincerely grateful to the financial support provided by the Intensification of Research in Priority Area (IRPA) fund for this research which was awarded to Dr. Amin Ismail. I also would like to express my gratitude for the tremendous help, support and contribution of the staff in the Department of Nutrition and Health Sciences, especially the laboratory staff, Pn. Siti Muskinah, En. Simon and Pn. Che Maznah for their technical advice and materials provision. Not to forget Prof. Jinap Selamat for every single equipment used for this project and each single personnel in cocoa lab, especially Pak Misnawi who had given very useful advise and guidance for this project to be a successful one.... Vlll
I also wish to express my deepest appreciation to my beloved parents, my family, Pn. Siti Muskinah and family, Polar Group members and my best friend, Bani Mat Wajib, who have given me encouragement and moral support in everyway during completion of this project Last but not least, I would also like to thank everyone who has contributed in making this project a reality.
I certify that an Examination Committee met on 25Lh April, 2005 to conduct the final examination of Ruzaidi Azli Mohd Mokhtar on his Master of Science thesis entitled "Effects of Ethanolic Extract of Cocoa on Blood Glucose and Lipid Profile in Streptozotocin-induced Diabetic Rats" in accordance with Universiti Pertanian Malaysia (Higher degree) act 1980 and Universiti Pertanian Malaysia (Higher degree) Regulation 198 1. The Committee recommends that the candidate be awarded the relevant degree. Members of the Examination Committee are as follows: ASMAH RAHMAT, PhD Associate Professor Faculty of Medicine and Health Sciences Universiti Putra Malaysia (Chairman) NORHAIZAN MOHD ESA, PhD Lecturer Faculty of Medicine and Health Sciences Universiti Putra Malaysia (Member) MOHD ROSLAN SULAIMAN, PhD Associate Professor Faculty of Medicine and Health Sciences Universiti Putra Malaysia (Member) AYUB MOHD YATIM, PhD Associate Professor Faculty of Science and Technology Universiti Kebangsaan Malaysia (Independent Examiner) Date: 2 1 JUL 2005
This thesis submitted to the Senate of Universiti Putra Malaysia and has been accepted as fulfillment of the requirements for the degree of Master of Science. The members of Supervisory Committee are as follows: AMIN ISMAIL, PhD Lecturer Faculty of Medicine and Health Sciences Universiti Putra Malaysia (Chairman) MUHA JIR HAMID, PhD Lecturer Faculty of Biotechnology and Bimolecular Sciences Universiti Putra Malaysia (Member) NAWALYAH ABDUL GHANI, M.SC. Lecturer Faculty of Medicine and Health Sciences Universiti Putra Malaysia (Member) AINI IDERIS, PhD Professor/Dean School of Graduate Studies Universiti Putra Malaysia Date: 1 \ AUG 2005
DECLARATION I hereby declare that the thesis is based on my original work except for quotations and citations which have been duly acknowledged. I also declare that it has not been previously or concurrently submitted for any other degree at Universiti Putra Malaysia or other institutions. RUZAIDI AZLI MOHD MOKHTAR Date: l't /6 /XQ< xii
TABLE OF CONTENTS Page DEDICATION ABSTRACT ABSTRAK ACKNOWLEDGEMENTS APPROVAL DECLARATION LIST OF TABLES LIST OF FIGURES LIST OF ABBREVIATIONS 11 iii v... Vlll X xii xv xvi xviii CHAPTER I INTRODUCTION Introduction Statement of Problem LITERATURE REVIEW Medicinal Aspects of Cocoa Technological Aspects of Cocoa Composition of Cocoa Beans Polyphenols in Cocoa Beans Cocoa Polyphenols on Degenerative Diseases Diabetes Cancer Cardiovascular Disease Antioxidants Diabetes Mellitus Lipid Metabolism in Diabetes Complications Treatments Insulin Receptor and Mechanism of Action MATERIALS AND METHODS Materials Source of Cocoa Beans Chemicals Methods Preparation of Cocoa Beans... Xlll
Preparation of Ethanolic Extract Determination of Polyphenol Content Animal Study In-vitro Study Statistical Analysis RESULTS Total Polyphenol and Procyanidin Content Total Polyphenol Content Procyanidin Composition Animal Study Effect of Cocoa Extract on Food Intake Effect of Cocoa Extract on Body Weight Effect of Cocoa Extract on Glucose Levels Effect of Cocoa Extract on Lipid Profiles Effect of Cocoa Extract on Insulin Secretion DISCUSSION Total Polyphenol Content and Procyanidin Composition Effect of Cocoa Extract on Food Intake, Body Weight and Glucose Levels Effect of Cocoa Extract on Lipid Profiles Effect of Cocoa Extract on Insulin Secretion CONCLUSIONS AND RECOMMENDATIONS Conclusions Recommendations REFERENCES APPENDICES AWARDS AND PUBLICATIONS BIODATA OF THE AUTHOR xiv
LIST OF TABLES Table 1 Epicatechin (EC) in fermented cocoa beans from different countries Mineral content of cocoa and cocoa products Main classes of phenolic compounds in plants Dietary recommendation for people with diabetes Composition of experimental diet Total polyphenol content in cocoa beans extracted with different concentration of ethanol Effect of CE on food intakes Effect of CE on body weight in rats (first batch) Effect of CE on body weight in rats (second batch) Effect of CE on body weight in rats (protective role of CE against STZ) Page 15
LIST OF FIGURES Figure Structure of common polyphenols found in cocoa Structure of human insulin Structure of insulin receptor Mechanism of glucose uptake in adipose cells Experiment design of animal study Experiment design of animal study (protective effect study) HPLC analysis of purified cocoa procyanidins Monomer, dimmer and tetramer in CE using normal-phase HPLC Body weight change pattern in normal rats (first batch) Body weight change pattern in diabetic rats (first batch) Body weight change pattern in normal rats (second batch) Body weight change pattern in diabetic rats (second batch) Body weight change pattern in control rats and those pretreated with CE Blood glucose profiles in normal rats fed with CE (first batch) Blood glucose profiles in diabetic rats fed with CE (first batch) Plasma glucose levels of normal rats fed with CE (first batch) Plasma glucose levels of diabetic rats fed with CE (first batch) Blood glucose profiles in normal rats fed with CE (second batch) Blood glucose profiles in diabetic rats fed with CE (second batch) Plasma glucose levels of normal rats fed with CE (second batch) Plasma glucose levels of diabetic rats fed with CE (second batch) Plasma glucose levels of diabetic rats pretreated with CE Plasma total cholesterol levels of normal rats fed with CE (first batch) Plasma total cholesterol levels of diabetic rats fed with CE (first batch) Plasma HDL-cholesterol levels of normal rats fed with CE (first batch) Plasma HDL-cholesterol levels of diabetic rats fed with CE (first batch) Plasma LDL-cholesterol levels of normal rats fed with CE (first batch) Page 20 44 47 48 56 60 70 70 76 77 80 81 84 xvi
28 Plasma LDL-cholesterol levels of diabetic rats fed with CE (first batch) 29 Plasma triglyceride levels of normal rats fed with CE (first batch) 30 Plasma triglyceride levels of diabetic rats fed with CE (first batch) 31 Plasma total cholesterol levels of normal rats fed with CE (second batch) 32 Plasma total cholesterol levels of diabetic rats fed with CE 110 (second batch) 33 Plasma HDL-cholesterol levels of normal rats fed with CE 111 (second batch) 34 Plasma HDL-cholesterol levels of diabetic rats fed with CE (second batch) 35 Plasma LDL-cholesterol levels of normal rats fed with CE (second batch) 36 Plasma LDL-cholesterol levels of diabetic rats fed with CE (second batch) 37 Plasma triglyceride levels of normal rats fed with CE (second batch) 38 Plasma triglyceride levels of diabetic rats fed with CE (second batch) 39 Plasma total cholesterol levels of diabetic rats pretreated with CE 40 Plasma HDL-cholesterol levels of diabetic rats pretreated with CE 41 Plasma WL-cholesterol levels of diabetic rats pretreated with CE 42 Plasma triglyceride levels of diabetic rats pretreated with CE 43 The effect of CE on in-vitro insulin secretion from BRIN-BDll cell lines xvii
LIST OF ABBREVIATIONS ADA b. ~ CE cv CVD g g HDL Hl'Lc hr I.D. M)a kg KRB 1. LDL M min MOH ng nm PBS STZ vlv WHO wlv P1 Pm : American Diabetes Association : boiling point : cocoa extract : coefficient of variance : cardiovascular disease : gram : gravity (relative centrifugal force) : high density lipoprotein : High Performance Liquid Chromatography : hour : internal diameter : kilodalton : kilogram : Krebs-Ringer bicarbonate : liter : low density lipoprotein : molarity : milligram : milliliter : minute : Ministry of Health, Malaysia : nanogram : nanometer : phosphate buffer-saline : streptozotocin : volume/volume : World Health Organization : weightfvolume : microliter : micrometer xviii
CHAPTER I INTRODUCTION Diabetes mellitus is a serious and costly disease which is becoming increasingly common, especially in developing countries. It is a disease with major long-term implications, not only on the health and well-being of the affected individuals, but also on the costs incurred by the government. For example in Canada, the annual estimated cost of treating patients with diabetes mellitus is 9 billion dollars, while in the United States it is estimated to be near 132 billion dollars in 2002 in medical expenditures and lost productivity (Dawson et al., 2002; ADA, 2003). There are no available statistics on the cost of treating diabetic patients in Malaysia, but WHO (2002) estimated that direct health care costs of diabetic patients range from 2.5% to 15% of total annual budget, depending on local diabetes prevalence and the effectiveness of the treatment available. In the latest WHO estimation (WHO, 2004), there are over 171 million people worldwide who are afflicted with diabetes mellitus. Diabetic individuals can suffer from ketoacidosis, a serious acute complication, as well as chronic complications that affect essentially every organ system in the body, among them cardiovascular diseases, stroke, blindness, kidney failure, neurological dysfunction, necrosis and gangrene.
The discovery of insulin in 1921 revolutionized diabetes treatment and greatly reduced the acute complication of diabetes mellitus. As diabetics began to live longer, however, the chronic complications have taken over as the principal cause of morbidity and mortality. Advances in our understanding of the pathophysiology of diabetes in the past several decades have produced significant improvements in therapy. There are two main types of diabetes mellitus: type 1, which was previously known as insulin-dependent diabetes mellitus (IDDM) or juvenile-onset diabetes mellitus, and is caused by absolute insulin deficiency; and type 2 or non-insulin dependent diabetes mellitus (NIDDM) or maturity-onset diabetes mellitus, which occurs mainly in adulthood, is often associated with obesity, and results from a combination of insulin resistance and @-cell dysfunction, leading to relative insulin deficiency (Kahn, 2003). Although diabetes mellitus is a non-communicable disease, it is considered one of the five leading causes of death in the world. Recently, the search for appropriate hypoglycaernic agents has focused on plants used in traditional medicine, partly because of leads provided by traditional medicine to natural products that may be better treatments than currently used drugs (Lu et al., 2002; Jang et al., 2003; Kar et al., 2003). Drug such as sulphonylureas, lead to higher risk of hypoglycaemia, and metformin brings a higher risk of lactic acidosis (Shenfield, 2001). Due to the side effects of these drugs, many researches have been conducting studies on natural products derived from plants with potential antidiabetic activities (Kamtchouing et al., 1998; Jayakar and Suresh, 2003; Ladeji et al., 2003; Maghrani et al., 2003).
Besides the traditional medicinal plants, cocoa beans were thought to have fearsome magical powers by the Mayas and were carefully used by priests in rituals, religious ceremonies and healings. The Mayas used cocoa medicinally as a treatment for fever, coughs and to help dispel even discomfort during pregnancy. After the 1 6 ~ century conquest of Central America by Spain, Cortes introduced cocoa to Europe, where it was typically viewed as a healthy and nutritious beverage (Dillinger et al., 2000). Therefore, to evaluate the hypoglycaemic and hypocholesterolaemic effect of cocoa beans, this study was designed to test its effectiveness in reducing hyperglycaemia and hypercholesterolaemia based on in-vivo (animal) and in-vitro studies. Malaysian is one of the main cocoa-based products producer in the world and the biggest in Asia. However, our local cocoa-based markets are more prefer other cocoa beans (West African and Ghanian beans) due to some weaknesses in Malaysian beans quality (low cocoa aroma, astringent and bitter taste). One of the factors causing this is believed to be due to the high amount of the polyphenol substances. Recently, polyphenols have become intense focus of research interest because of their antioxidant capacity and possible beneficial health effects. Thus, this study uses the superiority of Malaysian beans in order to evaluate its potential beneficial health effects especially in diabetes mellitus. Historically, animal models have been used to screen out extracts, pure compounds or drugs, and obtain information to help in understanding health disorders and to test
the safety of these materials before putting them on the market. Scientists from around the world usually use animal model to study the way the disease progresses, and factors that are important to the disease process. Animal models are also used to study the treatment of diseases before it can be applied to humans. Usually, after animal study is performed, it will be followed up with additional laboratory studies using cell culture (in-vitro model) for result confirmation. Streptozotocin (STZ)-induced hyperglycaemia in rats have been described as a useful experimental model to study the activity of antidiabetic agents with or without insulin (LeDoux et al., 1986). A range of STZ doses provide a wider range compared to alloxan, as an inducer of hyperglycaemia. The frequently-used single intravenous or intraperitoneal dose in adult rats to induce diabetes mellitus type 1 is between 40 and 60 mglkg body weight (Pepato et al., 2001; McAnuff et al., 2002), but higher doses are also used (Ladeji et al., 2003). In this study, two different batches of animal fed with cocoa extract (CE) via two different techniques were used: (1) CE mixed with purified diet, powder form and (2) forced-fed CE suspended in liquid solution. The aim of the experiment was to study the hypoglycaemic and hypocholesterolaemic effect of Malaysian cocoa extract. The rationale of using two different feeding techniques is to ensure the results derived from the second batch (force-feeding) supported the findings of the first batch (free excess to diet containing CE). In addition, for the first batch study, actual amount of food intake cannot be measured due to a lot of food powder being spilled. Furthermore, the food intake varied from one rat to another and because of that, it is difficult to measure the
exact polyphenol intake of the rats. Therefore, in order to make sure the exact doses of CE were taken by the rats, force-feeding using intubation needle (second batch) was designed to improve the effectiveness of this first batch design. The search for a safer and more effective compound in protecting the p-cells from inflammatory destruction by ST2 is still being done. Several compounds such as metallothionein, nicotinamide, glucose and (-)epicatechin have been reported to inhibit the diabetonic action of streptozotocin or alloxan (Kamtchouing et al., 1998; Yang and Cherian, 1994). Khalid (2002) found that palm Vitee (palm oil Vitamin E) has a protective property against the toxic inflammation caused by single dose STZ administration. Thus, this study was also designed to evaluate the protective action of CE against the destruction of insulin-producing p-cells of the pancreas in STZinduced diabetic rats. To understand the possible mechanisms by which CE improves hyperglycaemia, the effect of CE on insulin secretion by insulin-secreting cells was investigated. Insulinsecreting cell lines have provided useful systems for the study of pancreatic P-cell function. The BRIN-BD1 1 cell line is a clonal glucose responsive insulin-secreting cell line which is responsive to a range of pharmacological modulators of insulin secretion. Most of the available cell lines exhibit glucose insensitivity or moderate. - responsiveness to sub-physiological concentrations of glucose (Newgard, 1994). Thus, this study utilizes this cell line superiority to examine the effects of CE upon insulin secretion alone without the presence of glucose.